Synthesis of compounds useful as modulators of amyloid-beta production

ABSTRACT

As described herein, the present invention provides methods for preparing compounds useful for treating or lessening the severity of a neurodegenerative disorder. The present invention also provides methods of treating or lessening the severity of such disorders wherein said method comprises administering to a patient a compound of the present invention, or composition thereof. Said method is useful for treating or lessening the severity of, for example, Alzheimer&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 60/860,185, filed Nov. 20, 2006, the entirety of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to compounds that modulate amyloid-betapeptide production, processes for their preparation, and uses thereof.

BACKGROUND OF THE INVENTION

The central role of the long form of amyloid beta-peptide, in particularAβ(1-42), in Alzheimer's disease has been established through a varietyof histopathological, genetic and biochemical studies. See Selkoe, D J,Physiol. Rev. 2001, 81:741-766, Alzheimer's disease: genes, proteins,and therapy, and Younkin S G, J Physiol Paris. 1998, 92:289-92, The roleof A beta 42 in Alzheimer's disease. Specifically, it has been foundthat deposition in the brain of Aβ(1-42) is an early and invariantfeature of all forms of Alzheimer's disease. In fact, this occurs beforea diagnosis of Alzheimer's disease is possible and before the depositionof the shorter primary form of A-beta, Aβ(1-40). See Parvathy S, et al.Arch Neurol. 2001, 58:2025-32, Correlation between Abetax-40-,Abetax-42-, and Abetax-43-containing amyloid plaques and cognitivedecline. Further implication of Aβ(1-42) in disease etiology comes fromthe observation that mutations in presenilin (gamma secretase) genesassociated with early onset familial forms of Alzheimer's diseaseuniformly result in increased levels of Aβ(1-42). See Ishii K, et al.Neurosci Lett. 1997, 228:17-20, Increased A beta 42(43)-plaquedeposition in early-onset familial Alzheimer's disease brains with thedeletion of exon 9 and the missense point mutation (H163R) in the PS-1gene. Additional mutations in the amyloid precursor protein APP raisetotal Aβ and in some cases raise Aβ(1-42) alone. See Kosaka T, et al.Neurology, 48:741-5, The beta APP717 Alzheimer mutation increases thepercentage of plasma amyloid-beta protein ending at A beta42(43).Although the various APP mutations may influence the type, quantity, andlocation of Aβ deposited, it has been found that the predominant andinitial species deposited in the brain parenchyma is long Aβ (Mann). SeeMann D M, et al. Am J Pathol. 1996, 148:1257-66, Predominant depositionof amyloid-beta 42(43) in plaques in cases of Alzheimer's disease andhereditary cerebral hemorrhage associated with mutations in the amyloidprecursor protein gene.

In early deposits of Aβ, when most deposited protein is in the form ofamorphous or diffuse plaques, virtually all of the Aβ is of the longform. See Gravina S A, et al. J Biol Chem, 270:7013-6, Amyloid betaprotein (A beta) in Alzheimer's disease brain. Biochemical andimmunocytochemical analysis with antibodies specific for forms ending atA beta 40 or A beta 42(43); Iwatsubo T, et al. Am J Pathol. 1996,149:1823-30, Full-length amyloid-beta (1-42(43)) and amino-terminallymodified and truncated amyloid-beta 42(43) deposit in diffuse plaques;and Roher A E, et al. Proc Natl Acad Sci USA. 1993, 90:10836-40,beta-Amyloid-(1-42) is a major component of cerebrovascular amyloiddeposits: implications for the pathology of Alzheimer disease. Theseinitial deposits of Aβ(1-42) then are able to seed the furtherdeposition of both long and short forms of Aβ. See Tamaoka A, et al.Biochem Biophys Res Commun. 1994, 205:834-42, Biochemical evidence forthe long-tail form (A beta 1-42/43) of amyloid beta protein as a seedmolecule in cerebral deposits of Alzheimer's disease.

In transgenic animals expressing Aβ, deposits were associated withelevated levels of Aβ(1-42), and the pattern of deposition is similar tothat seen in human disease with Aβ(1-42) being deposited early followedby deposition of Aβ(1-40). See Rockenstein E, et al. J Neurosci Res.2001, 66:573-82, Early formation of mature amyloid-beta protein depositsin a mutant APP transgenic model depends on levels of Abeta(1-42); andTerai K, et al. Neuroscience 2001, 104:299-310, beta-Amyloid deposits intransgenic mice expressing human beta-amyloid precursor protein have thesame characteristics as those in Alzheimer's disease. Similar patternsand timing of deposition are seen in Down's Syndrome patients in whichAβ expression is elevated and deposition is accelerated. See Iwatsubo T,et al. Ann Neurol. 1995, 37:294-9, Amyloid beta protein (A beta)deposition: A beta 42(43) precedes A beta 40 in Down syndrome.

Accordingly, selective lowering of Aβ(1-42) thus emerges as adisease-specific strategy for reducing the amyloid forming potential ofall forms of Aβ, slowing or stopping the formation of new deposits ofAβ, inhibiting the formation of soluble toxic oligomers of Aβ, andthereby slowing or halting the progression of neurodegeneration.

SUMMARY OF THE INVENTION

As described herein, the present invention provides methods forpreparing compounds useful as modulators of amyloid-beta production.Such compounds are useful for treating or lessening the severity of aneurodegenerative disorder. The present invention also providesintermediates useful in carrying out such synthetic methods.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION 1. GeneralDescription of Compounds of the Invention

Methods and intermediates of the present invention are useful forpreparing compounds as described in, e.g. U.S. patent application Ser.No. 11/434,726, filed May 16, 2006, published as US 20070010503, in thename of Findeis et al., the entirety of which is incorporated herein byreference. In certain embodiments, the present compounds are generallyprepared according to Scheme I set forth below:

wherein each variable is as defined below and described in variousembodiments below and herein.

According to one embodiment, the present invention provides a method forpreparing a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   each of Ring A, Ring B, Ring C, Ring D, and Ring E is independently    saturated, partially unsaturated or aromatic;-   G is S, CH₂, NR, or O;-   R¹ and R² are each independently halogen, R, OR, a suitably    protected hydroxyl group, SR, a suitably protected thiol group,    N(R), or a suitably protected amino group, or R¹ and R² are taken    together to form a 3-7 membered saturated, partially unsaturated, or    aryl ring having 0-2 heteroatoms independently selected from    nitrogen, oxygen, or sulfur;-   each R is independently hydrogen, an optionally substituted C₁₋₆    aliphatic group, or an optionally substituted 3-8 membered    saturated, partially unsaturated, or aryl ring having 0-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    wherein:    -   two R on the same nitrogen atom are optionally taken together        with said nitrogen atom to form a 3-8 membered saturated,        partially unsaturated, or aryl ring having 1-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur;-   n is 0-2;-   R³, R⁴, R⁷, and R⁸ are each independently selected from halogen, R,    OR, a suitably protected hydroxyl group, SR, a suitably protected    thiol group, SO₂R, OSO₂R, N(R), a suitably protected amino group,    NR(CO)R, NR(CO)(CO)R, NR(CO)N(R)₂, NR(CO)OR, (CO)OR, O(CO)R,    (CO)N(R)₂, or O(CO)N(R)₂;-   m is 0-2;-   R⁵ is T-C(R′)₃, T-C(R′)₂C(R″)₃, R, OR, a suitably protected hydroxyl    group, SR, a suitably protected thiol group, SO₂R, OSO₂R, N(R), a    suitably protected amino group, NR(CO)R, NR(CO)(CO)R, NR(CO)N(R)₂,    NR(CO)OR, (CO)OR, O(CO)R, (CO)N(R)₂, or O(CO)N(R)₂, or:-   each T is independently a valence bond or an optionally substituted    straight or branched, saturated or unsaturated, C₁₋₆ alkylidene    chain wherein up to two methylene units of T are optionally and    independently replaced by —O—, —N(R)—, —S—, —C(O)—, —S(O)—, or    —S(O)₂—;-   each R′ and R″ is independently selected from R, OR, SR, SO₂R,    OSO₂R, N(R), NR(CO)R, NR(CO)(CO)R, NR(CO)N(R)₂, NR(CO)OR, (CO)OR,    O(CO)R, (CO)N(R)₂, or O(CO)N(R)₂;-   R⁹ and R^(9′) are each independently selected from halogen, R, OR,    SR, or N(R)₂, or R¹ and R² are taken together to form a 3-7 membered    saturated, partially unsaturated, or aryl ring having 0-2    heteroatoms independently selected from nitrogen, oxygen, or sulfur;-   Q is a valence bond or an optionally substituted straight or    branched, saturated or unsaturated, C₁₋₆ alkylidene chain wherein up    to two methylene units of Q are optionally and independently    replaced by —O—, —N(R)—, —S—, —C(O)—, —S(O)—, or —S(O)₂—; and-   R¹⁰ is R, a suitably protected hydroxyl group, a suitably protected    thiol group, a suitably protected amino group, an optionally    substituted 3-8 membered saturated, partially unsaturated, or aryl    monocyclic ring having 0-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, an optionally substituted 8-10 membered    saturated, partially unsaturated, or aryl bicyclic ring having 0-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    a detectable moiety, a polymer residue, a peptide, or a    sugar-containing or sugar-like moiety,    comprising the steps of:    (a) providing a compound of formula II:

and(b) converting the compound of formula II to the compound of formula I.

2. Definitions

Compounds of this invention include those described generally above, andare further illustrated by the embodiments, sub-embodiments, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry,” Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

As defined generally above, each of Ring A, Ring B, Ring C, Ring D, andRing E is independently saturated, partially unsaturated or aromatic. Itwill be appreciated that compounds of the present invention arecontemplated as chemically feasible compounds. Accordingly, it will beunderstood by one of ordinary skill in the art that when any of Ring A,Ring B, Ring C, Ring D, and Ring E is unsaturated, then certainsubstituents on that ring will be absent in order to satisfy generalrules of valency. For example, if Ring D is unsaturated at the bondbetween Ring D and Ring C, then R⁸ and R³ will be absent. Allcombinations of saturation and unsaturation of any of Ring A, Ring B,Ring C, Ring D, and Ring E are contemplated by the present invention.Thus, in order to satisfy general rules of valency, and depending on thedegree of saturation or unsaturation of any of Ring A, Ring B, Ring C,Ring D, and Ring E, the requisite presence or absence of each of R¹, R²,R³, R⁴, R⁵, R⁷, R⁸, R⁹, R^(9′), and QR¹⁰ is contemplated accordingly.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted,”whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds.

The term “stable,” as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and preferably their recovery, purification, anduse for one or more of the purposes disclosed herein. In someembodiments, a stable compound or chemically feasible compound is onethat is not substantially altered when kept at a temperature of 40° C.or less, in the absence of moisture or other chemically reactiveconditions, for at least a week.

The term “aliphatic” or “aliphatic group,” as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-20 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-6aliphatic carbon atoms. In yet other embodiments aliphatic groupscontain 1-4 aliphatic carbon atoms. In some embodiments,“cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to amonocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule wherein any individual ring in said bicyclic ringsystem has 3-7 members. Suitable aliphatic groups include, but are notlimited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl. In other embodiments, analiphatic group may have two geminal hydrogen atoms replaced with oxo (abivalent carbonyl oxygen atom═O), or a ring-forming substituent, such as—O-(straight or branched alkylene or alkylidene)-O— to form an acetal orketal.

In certain embodiments, exemplary aliphatic groups include, but are notlimited to, ethynyl, 2-propynyl, 1-propenyl, 2-butenyl, 1,3-butadienyl,2-pentenyl, vinyl (ethenyl), allyl, isopropenyl, methyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, sec-pentyl, neo-pentyl, tert-pentyl, cyclopentyl, hexyl,isohexyl, sec-hexyl, cyclohexyl, 2-methylpentyl, tert-hexyl,2,3-dimethylbutyl, 3,3-dimethylbutyl, 1,3-dimethylbutyl, and2,3-dimethylbut-2-yl.

The term “heterocycle,” “heterocyclyl,” “heterocycloaliphatic,” or“heterocyclic” as used herein means non-aromatic, monocyclic, bicyclic,or tricyclic ring systems in which one or more ring members is anindependently selected heteroatom. In some embodiments, the“heterocycle,” “heterocyclyl,” “heterocycloaliphatic,” or “heterocyclic”group has three to fourteen ring members in which one or more ringmembers is a heteroatom independently selected from oxygen, sulfur,nitrogen, or phosphorus, and each ring in the system contains 3 to 7ring members.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl).

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

The term “alkoxy,” or “thioalkyl,” as used herein, refers to an alkylgroup, as previously defined, attached to the principal carbon chainthrough an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains 3 to 7 ring members. The term“aryl” may be used interchangeably with the term “aryl ring.” The term“aryl” also refers to heteroaryl ring systems as defined hereinbelow.

The term “heteroaryl,” used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy,” refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms, and whereineach ring in the system contains 3 to 7 ring members. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic.”

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) orheteroaryl (including heteroaralkyl and heteroarylalkoxy and the like)group may contain one or more substituents. Suitable substituents on theunsaturated carbon atom of an aryl or heteroaryl group are selected fromhalogen; N₃, CN, R^(o); OR^(o); SW); 1,2-methylene-dioxy;1,2-ethylenedioxy; phenyl (Ph) optionally substituted with R^(o); —O(Ph)optionally substituted with R^(o); (CH₂)₁₋₂(Ph), optionally substitutedwith R^(o); CH═CH(Ph), optionally substituted with R^(o); NO₂; CN;N(RO₂; NR^(o)C(O)R^(o); NR^(o)C(O)N(RO₂; NR^(o)CO₂R^(o);—NR^(o)NR^(o)C(O)R^(o); NR^(o)NR^(o)C(O)N(RO₂; NR^(o)NR^(o)CO₂R^(o);C(O)C(O)R^(o); C(O)CH₂C(O)R^(o); CO₂R^(o); C(O)R^(o); C(O)N(RO₂;OC(O)N(R^(o) ₂; S(O)₂R^(o); S)O₂N(R^(o) ₂; S(O)R^(o); NR^(o)SO₂N(R^(o)₂; NR^(o)SO₂R^(o); C(═S)N(RO₂; C(═NH)—N(RO₂; or (CH₂)₀₋₂NHC(O)R^(o)wherein each independent occurrence of R^(o) is selected from hydrogen,optionally substituted C₁₋₆ aliphatic, an unsubstituted 5-6 memberedheteroaryl or heterocyclic ring, phenyl, O(Ph), or CH₂(Ph), or,notwithstanding the definition above, two independent occurrences ofR^(o), on the same substituent or different substituents, taken togetherwith the atom(s) to which each R^(o) group is bound, form a 3-8 memberedcycloalkyl, heterocyclyl, aryl, or heteroaryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur.Optional substituents on the aliphatic group of R^(o) are selected fromN₃, CN, NH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halogen, C₁₋₄aliphatic, OH, O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄ aliphatic),O(haloC₁₋₄ aliphatic), or haloC_(i-4) aliphatic, wherein each of theforegoing C₁₋₄ aliphatic groups of R^(o) is unsubstituted.

An aliphatic or heteroaliphatic group or a non-aromatic heterocyclicring may contain one or more substituents. Suitable substituents on thesaturated carbon of an aliphatic or heteroaliphatic group, or of anon-aromatic heterocyclic ring are selected from those listed above forthe unsaturated carbon of an aryl or heteroaryl group and additionallyinclude the following: ═O, ═S, ═NNHR*, ═NN(R*)₂, ═NNHC(O)R*,═NNHCO₂(alkyl), ═NNHSO₂(alkyl), or ═NR*, where each R* is independentlyselected from hydrogen or an optionally substituted C₁₋₆ aliphatic.Optional substituents on the aliphatic group of R* are selected fromNH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halogen, C₁₋₄ aliphatic,OH, O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄ aliphatic), O(halo C₁₋₄aliphatic), or halo(C₁₋₄ aliphatic), wherein each of the foregoing C₁₋₄aliphatic groups of R* is unsubstituted.

Optional substituents on the nitrogen of a non-aromatic heterocyclicring are selected from R⁺, N(R⁺)₂, C(O)R⁺, CO₂R⁺, C(O)C(O)R⁺,C(O)CH₂C(O)R⁺, SO₂R⁺, SO₂N(R⁺)₂, C(═S)N(R⁺)₂, C(═NH)—N(R⁺)₂, orNR⁺SO₂R⁺; wherein R⁺is hydrogen, an optionally substituted C₁₋₆aliphatic, optionally substituted phenyl, optionally substituted O(Ph),optionally substituted CH₂(Ph), optionally substituted (CH₂)₁₋₂(Ph);optionally substituted CH═CH(Ph); or an unsubstituted 5-6 memberedheteroaryl or heterocyclic ring having one to four heteroatomsindependently selected from oxygen, nitrogen, or sulfur, or,notwithstanding the definition above, two independent occurrences of R⁺,on the same substituent or different substituents, taken together withthe atom(s) to which each R⁺ group is bound, form a 3-8-memberedcycloalkyl, heterocyclyl, aryl, or heteroaryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur.Optional substituents on the aliphatic group or the phenyl ring of R⁺are selected from NH₂, NH(C₁₋₄ aliphatic), N(C₁₋₄ aliphatic)₂, halogen,C₁₋₄ aliphatic, OH, O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄aliphatic), O(halo C₁₋₄ aliphatic), or halo(C₁₋₄ aliphatic), whereineach of the foregoing C₁₋₄ aliphatic groups of R⁺ is unsubstituted.

As detailed above, in some embodiments, two independent occurrences ofR^(o) (or R⁺, or any other variable similarly defined herein), are takentogether with the atom(s) to which each variable is bound to form a3-8-membered cycloalkyl, heterocyclyl, aryl, or heteroaryl ring having0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.Exemplary rings that are formed when two independent occurrences ofR^(o) (or R⁺, or any other variable similarly defined herein) are takentogether with the atom(s) to which each variable is bound include, butare not limited to the following: a) two independent occurrences ofR^(o) (or R⁺, or any other variable similarly defined herein) that arebound to the same atom and are taken together with that atom to form aring, for example, N(R^(o) ₂, where both occurrences of R^(o) are takentogether with the nitrogen atom to form a piperidin-1-yl,piperazin-1-yl, or morpholin-4-yl group; and b) two independentoccurrences of R^(o) (or R⁺, or any other variable similarly definedherein) that are bound to different atoms and are taken together withboth of those atoms to form a ring, for example where a phenyl group issubstituted with two occurrences of OR^(o)

these two occurrences of R^(o) are taken together with the oxygen atomsto which they are bound to form a fused 6-membered oxygen containingring:

It will be appreciated that a variety of other rings can be formed whentwo independent occurrences of R^(o) (or R⁺, or any other variablesimilarly defined herein) are taken together with the atom(s) to whicheach variable is bound and that the examples detailed above are notintended to be limiting.

As used herein, the term “detectable moiety” is used interchangeablywith the term “label” and relates to any moiety capable of beingdetected, e.g., primary labels and secondary labels. Primary labels,such as radioisotopes (e.g., ³²P, ³³P, ³⁵S, or ¹⁴C), mass-tags, andfluorescent labels are signal generating reporter groups which can bedetected without further modifications.

The term “secondary label” as used herein refers to moieties such asbiotin and various protein antigens that require the presence of asecond intermediate for production of a detectable signal. For biotin,the secondary intermediate may include streptavidin-enzyme conjugates.For antigen labels, secondary intermediates may include antibody-enzymeconjugates. Some fluorescent groups act as secondary labels because theytransfer energy to another group in the process of nonradiativefluorescent resonance energy transfer (FRET), and the second groupproduces the detected signal.

The terms “fluorescent label,” “fluorescent dye,” and “fluorophore” asused herein refer to moieties that absorb light energy at a definedexcitation wavelength and emit light energy at a different wavelength.Examples of fluorescent labels include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568,BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue,Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5),Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, and Texas Red-X.

The term “mass-tag” as used herein refers to any moiety that is capableof being uniquely detected by virtue of its mass using mass spectrometry(MS) detection techniques. Examples of mass-tags include electrophorerelease tags such asN-[3-[4′-[(p-Methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]isonipecoticAcid, 4′-[2,3,5,6-Tetrafluoro-4-(pentafluorophenoxyl)]methylacetophenone, and their derivatives. The synthesis and utility of thesemass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016,5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270.Other examples of mass-tags include, but are not limited to,nucleotides, dideoxynucleotides, oligonucleotides of varying length andbase composition, oligopeptides, oligosaccharides, and other syntheticpolymers of varying length and monomer composition. A large variety oforganic molecules, both neutral and charged (biomolecules or syntheticcompounds) of an appropriate mass range (100-2000 Daltons) may also beused as mass-tags.

The term “substrate,” as used herein refers to any material ormacromolecular complex to which a functionalized end-group of a compoundof the present invention can be attached. Examples of commonly usedsubstrates include, but are not limited to, glass surfaces, silicasurfaces, plastic surfaces, metal surfaces, surfaces containing ametallic or chemical coating, membranes (e.g., nylon, polysulfone,silica), micro-beads (e.g., latex, polystyrene, or other polymer),porous polymer matrices (e.g., polyacrylamide gel, polysaccharide,polymethacrylate), and macromolecular complexes (e.g., protein,polysaccharide).

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention.

Unless otherwise stated, all tautomeric forms of the compounds of theinvention are within the scope of the invention.

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as analytical tools or probes in biological assays.

3. Description of Exemplary Embodiments

As described generally above, the present invention provides a methodfor preparing a compound of formula I:

comprising the steps of:(a) providing a compound of formula II:

and(b) converting the compound of formula II to the compound of formula I.

One of ordinary skill in the art will recognize that the conversion of acompound of formula II to a compound of formula I is readilyaccomplished by an intramolecular dehydration reaction. Such dehydrationreactions are known to one of ordinary skill in the art and involve theloss of a water molecule to form a double bond.

In certain embodiments, the conversion of a compound of formula II to acompound of formula I is achieved by treatment of the compound offormula II with a suitable acid. As used herein, the phrase “suitableacid” refers to an organic or inorganic acid in an amount sufficient toaccomplish the dehydration of a compound of formula II to form acompound of formula I. Such suitable acids are well known in the art andinclude inorganic acids, e.g. hydrochloric acid, hydrobromic acid,phosphoric acid, nitric acid, sulfuric acid or perchloric acid, ororganic acids, e.g. acetic acid, oxalic acid, maleic acid, tartaricacid, citric acid, succinic acid, malonic acid, lower alkyl sulfonicacids or aryl sulfonic acids. In certain embodiments, the suitable acidis a Brønsted acid, such as hydrogen halides, carboxylic acids, sulfonicacids, sulfuric acid, and phosphoric acid. In certain embodiments, thesuitable acid is TFA.

The conversion of a compound of formula II to a compound of formula I isoptionally performed in the presence of a suitable solvent. A suitablesolvent is a single solvent or a solvent mixture that, in combinationwith the reagents, may facilitate the progress of the reactiontherebetween. The suitable solvent may solubilize one or more of thereaction components, or, alternatively, the suitable solvent mayfacilitate the agitation of a suspension of one or more of the reactioncomponents. Examples of suitable solvents useful in the presentinvention are a protic solvent, a halogenated hydrocarbon, an ether, anester, an aromatic hydrocarbon, a polar or a non-polar aprotic solvent,or any mixtures thereof. Such mixtures include, for example, mixtures ofprotic and non-protic solvents such as benzene/methanol/water;benzene/water; DME/water, and the like. In certain embodiments, thesuitable solvent is a polar aprotic solvent, such as an alcohol.

In certain embodiments, the suitable acid may also serve as the suitablesolvent.

In certain embodiments, the conversion of a compound of formula II to acompound of formula I is performed with optional heating.

As defined generally above, the G moiety of formulae I and II is S, CH₂,NR, or O. In certain embodiments, the G moiety of formulae I and II isO.

As defined generally above, R¹ and R² of formulae I and II are eachindependently halogen, R, OR, a suitably protected hydroxyl group, SR, asuitably protected thiol group, N(R)₂, or a suitably protected aminogroup, or R¹ and R² are taken together to form a 3-7 membered saturated,partially unsaturated, or aryl ring having 0-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, R¹and R² of formulae I and II are each independently R or OR. In otherembodiments, R¹ and R² of formulae I and II are each independently R,wherein R is hydrogen or an optionally substituted C₁₋₆ aliphatic group.According to another aspect of the present invention, R¹ and R² offormulae I and II are taken together to form a 3-6 membered saturated,partially unsaturated, or aryl ring having 0-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. Yet another aspect of thepresent invention provides a compound of formulae I and II wherein R¹and R² are taken together to form a 3-6 membered saturated carbocyclicring. In other embodiments, R¹ and R² of formula I are taken together toform a cyclopropyl ring.

In certain embodiments, the n moiety of formulae I and II is 0-1. Inother embodiments, the n moiety of formulae I and II is 1.

As defined generally above, the R⁵ group of formulae I and II is R⁵ isT-C(R′)₃, T-C(R′)₂C(R″)₃, R, OR, a suitably protected hydroxyl group,SR, a suitably protected thiol group, SO₂R, OSO₂R, N(R)₂, a suitablyprotected amino group, NR(CO)R, NR(CO)(CO)R, NR(CO)N(R)₂, NR(CO)OR,(CO)OR, O(CO)R, (CO)N(R)₂, or O(CO)N(R)₂, wherein each T isindependently a valence bond or an optionally substituted straight orbranched, saturated or unsaturated, C₁₋₆ alkylidene chain wherein up totwo methylene units of T are optionally and independently replaced by—O—, —N(R)—, —S—, —C(O)—, —S(O)—, or —S(O)₂—. In certain embodiments,each T is independently a valence bond or a straight or branched C₁₋₄alkylidene chain wherein one methylene unit of T is optionally replacedby —O—, —N(R)—, or —S—. In other embodiments, each T is independently avalence bond or a straight or branched C₁₋₄ alkylidene chain. In stillother embodiments, each T is a valence bond.

When the R⁵ group of formulae I and II is T-C(R′)₃ or T-C(R′)₂C(R″)₃,each R′ and R″ is independently selected from R, OR, SR, SO₂R, OSO₂R,N(R)₂, NR(CO)R, NR(CO)(CO)R, NR(CO)N(R)₂, NR(CO)OR, (CO)OR, O(CO)R,(CO)N(R)₂, or O(CO)N(R)₂. In certain embodiments, each R′ and R″ isindependently R, OR, OC(O)R, SR, or N(R)₂. In other embodiments, each R′and R″ is independently R, OR, or OC(O)R. Exemplary R′ and R″ groupsinclude hydrogen, CH₃, OH, and OC(O)CH₃.

As defined generally above, the R⁵ group of formulae I and II is, interalia, a suitably protected hydroxyl group, a suitably protected thiolgroup, or a suitably protected amino group. Hydroxyl protecting groupsare well known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, the entirety of which isincorporated herein by reference. Examples of suitably protectedhydroxyl groups of the R⁵ group of formulae I and II further include,but are not limited to, esters, allyl ethers, ethers, silyl ethers,alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of suchesters include formates, acetates, carbonates, and sulfonates. Specificexamples include formate, benzoyl formate, chloroacetate,trifluoroacetate, methoxyacetate, triphenylmethoxyacetate,p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate,4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetyl), crotonate,4-methoxy-crotonate, benzoate, p-benzylbenzoate,2,4,6-trimethylbenzoate, carbonates such as methyl, 9-fluorenylmethyl,ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl. Examples ofsuch silyl ethers include trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and othertrialkylsilyl ethers. Alkyl ethers include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allyl, andallyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers includeacetals such as methoxymethyl, methylthiomethyl,(2-methoxyethoxy)methyl, benzyloxymethyl,beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers.Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM),3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl.

Thiol protecting groups are well known in the art and include thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, theentirety of which is incorporated herein by reference. Suitablyprotected thiol groups of the R⁵ moiety of formulae I and II include,but are not limited to, disulfides, thioethers, silyl thioethers,thioesters, thiocarbonates, thiocarbamates, and the like. Examples ofsuch groups include, but are not limited to, alkyl thioethers, benzyland substituted benzyl thioethers, triphenylmethyl thioethers, andtrichloroethoxycarbonyl, to name but a few.

According to another aspect of the present invention, the R⁵ moiety offormulae I and II is a thiol protecting group that is removable underneutral conditions e.g. with AgNO₃, HgCl₂, and the like. Other neutralconditions include reduction using a suitable reducing agent. Suitablereducing agents include dithiothreitol (DTT), mercaptoethanol,dithionite, reduced glutathione, reduced glutaredoxin, reducedthioredoxin, substituted phosphines such as tris carboxyethyl phosphine(TCEP), and any other peptide or organic based reducing agent, or otherreagents known to those of ordinary skill in the art. According to yetanother aspect of the present invention, the R⁵ moiety of formulae I andII is a thiol protecting group that is “photocleavable”. Such suitablethiol protecting groups are known in the art and include, but are notlimited to, a nitrobenzyl group, a tetrahydropyranyl (THP) group, atrityl group, —CH₂SCH₃ (MTM), dimethylmethoxymethyl, or—CH₂—S—S-pyridin-2-yl. One of ordinary skill in the art would recognizethat many of the suitable hydroxylprotecting groups, as describedherein, are also suitable as thiol protecting groups.

In certain embodiments, the R⁵ group of formulae I and II is a suitablyprotected amino group. Amino protecting groups are well known in the artand include those described in detail in Protecting Groups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley &Sons, 1999, the entirety of which is incorporated herein by reference.Suitably protected amino groups of said R⁵ moiety further include, butare not limited to, aralkylamines, carbamates, cyclic imides, allylamines, amides, and the like. Examples of such groups includet-butyloxycarbonyl (BOC), ethyloxycarbonyl, methyloxycarbonyl,trichloroethyloxycarbonyl, allyloxycarbonyl (Alloc), benzyloxocarbonyl(CBZ), allyl, phthalimide, benzyl (Bn), fluorenylmethylcarbonyl (Fmoc),formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl,phenylacetyl, trifluoroacetyl, benzoyl, and the like. In certainembodiments, the amino protecting group of the R⁵ moiety is phthalimido.In still other embodiments, the amino protecting group of the R⁵ moietyis a tert-butyloxycarbonyl (BOC) group.

As defined generally above, the Q group of formulae I and II is avalence bond or an optionally substituted straight or branched,saturated or unsaturated, C₁₋₄ alkylidene chain wherein up to twomethylene units of Q are optionally and independently replaced by —O—,—N(R)—, —S—, —C(O)—, —S(O)—, or —S(O)₂—. In certain embodiments, Q is aan optionally substituted straight or branched, saturated orunsaturated, C₁₋₂ alkylidene chain wherein up to one methylene unit of Qis optionally replaced by —O—, —N(R)—, or —S—. In other embodiments, Qis —O—.

As defined generally above, the R¹⁰ group of formulae I and II is R, asuitably protected hydroxyl group, a suitably protected thiol group, asuitably protected amino group, an optionally substituted 3-8 memberedsaturated, partially unsaturated, or aryl monocyclic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, anoptionally substituted 8-10 membered saturated, partially unsaturated,or aryl bicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, a detectable moiety, a polymer residue, apeptide, or a sugar-containing group, or a sugar-like group.

In certain embodiments, the R¹⁰ group of formulae I and II is asugar-containing group. Such sugar-containing groups are well known toone of ordinary skill in the art and include those described in detailin “Essentials of Glycobiology” Edited by Varki, A., et al., Cold SpringHarbor Laboratory Press. Cold Spring Harbor, N.Y. 2002. In certainembodiments, the R¹⁰ group of formulae I and II is a glycoside.Exemplary R¹⁰ groups include arabinopyranosides and xylopyranosides. Incertain embodiments, the R¹⁰ group of formulae I and II is axylopyranoside. In certain embodiments, the R¹⁰ group of formulae I andII is an arabinopyranoside. In still other embodiments, the R¹⁰ group offormulae I and II is

According to another embodiment, the R¹⁰ group of formulae I and II is

Yet another embodiment provides a compound of formulae I and II whereinR¹⁰ is

According to another aspect of the present invention, the R¹⁰ group offormulae I and II is a sugar-mimetic. Such sugar-mimetics are well knownto one of ordinary skill in the art and include those described indetail in “Essentials of Glycobiology.” For example, sugar-mimeticgroups contemplated by the present invention include cyclitols and thelike. In certain embodiments, R¹⁰ is a cyclitol moiety, wherein saidcyclitol is a cycloalkane containing one hydroxyl group on each of threeor more ring atoms, as defined by IUPAC convention. In otherembodiments, such cyclitol moieties include inositols such asscyllo-inositol.

In addition, suitable sugar-like moieties of the R¹⁰ group of formulae Iand II include acyclic sugar groups. Such groups include linear alkylolsand erythritols, to name but a few. It will be appreciated that sugargroups can exist in either cyclic or acyclic form. Accordingly, acyclicforms of a sugar group are contemplated by the present invention as asuitable sugar-like moiety of the R¹⁰ group of formulae I and II.

In certain embodiments, the R¹⁰ group of formulae I and II is adetectable moiety. In other embodiments, the R¹⁰ group of formulae I andII is a fluorescent label, fluorescent dye, or fluorophore as definedherein, supra.

According to another aspect of the present invention, the R¹⁰ group offormulae I and II is a polymer residue. Polymer residues are well knownin the art and include those described in detail in “Chemistry ofProtein Conjugation and Cross-Linking” Shan S. Wong, CRC Press. BocaRaton, Fla. 1991. Suitable polymer residues of the R¹⁰ group of formulaeI and II include poly(alkylene oxides), such as PEG, poly(amino acids),and other polymer residues capable of conjugation to a compound of thepresent invention.

As defined generally above, the R¹⁰ group of formulae I and II is, interalia, a suitably protected hydroxyl group, a suitably protected thiolgroup, or a suitably protected amino group. Hydroxyl protecting groupsare well known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, the entirety of which isincorporated herein by reference. Examples of suitable hydroxylprotecting groups of the R¹⁰ group of formulae I and II further include,but are not limited to, esters, allyl ethers, ethers, silyl ethers,alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of suchesters include formates, acetates, carbonates, and sulfonates. Specificexamples include formate, benzoyl formate, chloroacetate,trifluoroacetate, methoxyacetate, triphenylmethoxyacetate,p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate,4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetyl), crotonate,4-methoxy-crotonate, benzoate, p-benzylbenzoate,2,4,6-trimethylbenzoate, carbonates such as methyl, 9-fluorenylmethyl,ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl. Examples ofsuch silyl ethers include trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and othertrialkylsilyl ethers. Alkyl ethers include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allyl, andallyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers includeacetals such as methoxymethyl, methylthiomethyl,(2-methoxyethoxy)methyl, benzyloxymethyl,beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers.Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM),3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl.

Thiol protecting groups are well known in the art and include thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, theentirety of which is incorporated herein by reference. Suitable thiolprotecting groups of the R¹⁰ moiety of formulae I and II include, butare not limited to, disulfides, thioethers, silyl thioethers,thioesters, thiocarbonates, thiocarbamates, and the like. Examples ofsuch groups include, but are not limited to, alkyl thioethers, benzyland substituted benzyl thioethers, triphenylmethyl thioethers,trichloroethoxycarbonyl, to name but a few.

According to another aspect of the present invention, the R¹⁰ moiety offormulae I and II is a thiol protecting group that is removable underneutral conditions e.g. with AgNO₃, HgCl₂, and the like. Other neutralconditions include reduction using a suitable reducing agent. Suitablereducing agents include dithiothreitol (DTT), mercaptoethanol,dithionite, reduced glutathione, reduced glutaredoxin, reducedthioredoxin, substituted phosphines such as tris carboxyethyl phosphine(TCEP), and any other peptide or organic based reducing agent, or otherreagents known to those of ordinary skill in the art. According to yetanother aspect of the present invention, the R¹⁰ moiety of formulae Iand II is a thiol protecting group that is “photocleavable.” Suchsuitable thiol protecting groups are known in the art and include, butare not limited to, a nitrobenzyl group, a tetrahydropyranyl (THP)group, a trityl group, —CH₂SCH₃ (MTM), dimethylmethoxymethyl, or—CH₂—S—S-pyridin-2-yl. One of ordinary skill in the art would recognizethat many of the suitable hydroxyl protecting groups, as describedherein, are also suitable as thiol protecting groups.

In certain embodiments, the R¹⁰ group of formulae I and II is a suitablyprotected amino group. Amino protecting groups are well known in the artand include those described in detail in Protecting Groups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley &Sons, 1999, the entirety of which is incorporated herein by reference.Suitable amino protecting groups of said R¹⁰ moiety further include, butare not limited to, aralkylamines, carbamates, cyclic imides, allylamines, amides, and the like. Examples of such groups includet-butyloxycarbonyl (BOC), ethyloxycarbonyl, methyloxycarbonyl,trichloroethyloxycarbonyl, allyloxycarbonyl (Alloc), benzyloxocarbonyl(CBZ), allyl, phthalimide, benzyl (Bn), fluorenylmethylcarbonyl (Fmoc),formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl,phenylacetyl, trifluoroacetyl, benzoyl, and the like. In certainembodiments, the amino protecting group of the R¹⁰ moiety isphthalimido. In still other embodiments, the amino protecting group ofthe R¹⁰ moiety is a tert-butyloxycarbonyl (BOC) group.

As described generally above, the present invention provides a methodfor preparing a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein each variable isdefined above and in classes and subclasses described above and herein.In certain embodiments, the present invention provides a method forpreparing a compound of formula I having the stereochemistry as depictedin formula I-a:

or a pharmaceutically acceptable salt thereof, wherein each variable isdefined above and in classes and subclasses described above and hereinfor compounds of formula I.

In certain embodiments, the present invention provides a method forpreparing a compound of formula I, as described herein, wherein saidcompound is of formula I-b:

or a pharmaceutically acceptable salt thereof, wherein each variable isdefined above and in classes and subclasses described above and hereinfor compounds of formula I.

In other embodiments, the present invention provides a method forpreparing a compound of formula I, as described herein, wherein saidcompound is of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein each variable isdefined above and in classes and subclasses described above and hereinfor compounds of formula I.

As defined generally above, each of Ring A, Ring B, Ring C, Ring D, andRing E is independently saturated, partially unsaturated or aromatic. Incertain embodiments, the present invention provides a method ofpreparing a compound of formula I wherein Ring B is unsaturated and R¹and R² are absent, thus forming a compound of formula I-d:

or a pharmaceutically acceptable salt thereof, wherein each variable isdefined above and in classes and subclasses described above and hereinfor compounds of formula I.

In certain embodiments, the n group of formula I-d is 0-1 and the Ggroup of formula I-d is oxygen.

In other embodiments, the present invention provides a method forpreparing compound of formula I-i:

or a pharmaceutically acceptable salt thereof, wherein:

-   Q is a valence bond or an optionally substituted straight or    branched, saturated or unsaturated, C₁₋₆ alkylidene chain wherein up    to two methylene units of Q are optionally and independently    replaced by —O—, —N(R)—, —S—, —C(O)—, —S(O)—, or —S(O)₂—;-   R¹⁰ is R, a suitably protected hydroxyl group, a suitably protected    thiol group, a suitably protected amino group, an optionally    substituted 3-8 membered saturated, partially unsaturated, or aryl    monocyclic ring having 0-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, an optionally substituted 8-10 membered    saturated, partially unsaturated, or aryl bicyclic ring having 0-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    a detectable moiety, a polymer residue, a peptide, or a    sugar-containing or sugar-like moiety; and-   R is hydrogen, an optionally substituted C₁₋₆ aliphatic group, or an    optionally substituted 3-8 membered saturated, partially    unsaturated, or aryl ring having 0-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, wherein:    -   two R on the same nitrogen atom are optionally taken together        with said nitrogen atom to form a 3-8 membered saturated,        partially unsaturated, or aryl ring having 1-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur,        comprising the steps of:        (a) providing a compound of formula I-ii:

and(b) converting compound I-ii to compound I-i.

In other embodiments, the present invention provides a method forpreparing compound of formula I-iii:

or a pharmaceutically acceptable salt thereof, wherein:

-   Q is a valence bond or an optionally substituted straight or    branched, saturated or unsaturated, C₁₋₆ alkylidene chain wherein up    to two methylene units of Q are optionally and independently    replaced by —O—, —N(R)—, —S—, —C(O)—, —S(O)—, or —S(O)₂—;-   R¹⁰ is R, a suitably protected hydroxyl group, a suitably protected    thiol group, a suitably protected amino group, an optionally    substituted 3-8 membered saturated, partially unsaturated, or aryl    monocyclic ring having 0-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, an optionally substituted 8-10 membered    saturated, partially unsaturated, or aryl bicyclic ring having 0-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    a detectable moiety, a polymer residue, a peptide, or a    sugar-containing or sugar-like moiety; and-   R is hydrogen, an optionally substituted C₁₋₆ aliphatic group, or an    optionally substituted 3-8 membered saturated, partially    unsaturated, or aryl ring having 0-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, wherein:    -   two R on the same nitrogen atom are optionally taken together        with said nitrogen atom to form a 3-8 membered saturated,        partially unsaturated, or aryl ring having 1-4 heteroatoms        independently selected from nitrogen, oxygen, or sulfur,        comprising the steps of:        (a) providing a compound of formula I-iv:

and(b) converting compound I-iv to compound I-iii.

4. General Methods of Providing the Present Compounds

The compounds of formula II of this invention may be prepared orisolated in general by synthetic and/or semi-synthetic methods known tothose skilled in the art for analogous compounds and by methodsdescribed in detail in the Examples, below.

In certain embodiments, methods of the present invention for convertinga compound of formula II to a compound of formula I are performed usingan isolated compound of formula II. Isolated compounds of formula II maybe provided by purification/separation from an extract of black cohoshroot. Alternatively, isolated compounds of formula II may be provided bytotal synthesis or semi-synthesis. In other embodiments, the conversionof a compound of formula II to a compound of formula I is performedusing an extract of black cohosh root that is enriched in a compound offormula II. Such isolation methods and enriched extracts are describedin more detail below.

Isolation of Components

Certain compounds of the present invention were isolated from blackcohosh root, also known as cimicifuga racemosa or actaea racemosa, andthe structure of these compounds elucidated. Commercial extracts,powders, and capsules of black cohosh root are available for treating avariety of menopausal and gynecological disorders. However, it has beensurprisingly found that certain compounds present in black cohosh rootare useful for modulating and/or inhibiting amyloid-beta peptideproduction. In particular, certain compounds have been isolated fromblack cohosh root and identified, wherein these compounds are useful formodulating and/or inhibiting amyloid-beta peptide production especiallyamyloid-beta peptide (1-42). Such compounds are encompassed by formulaeI and II. These compounds may be isolated and utilized in a formsubstantially free of other compounds normally found in the root.Alternatively, an extract may be prepared from the root wherein saidextract is enriched in a compound of the present invention.

As described above and herein, certain compounds useful in methods ofthe present invention are isolated from standard extracts of cultivatedor wild-grown black cohosh roots and rhizomes. It is also contemplatedthat the present compounds may also be isolated from plant root tissuegrown in culture or from the culture medium of the culture plant tissue.Such methods of growing plant root tissue in culture are well known toone of ordinary skill in the art and include those described in HairyRoots, Culture and Applications, edited by Pauline M. Doran, publishedby Harwood Academic Publishers, Amsterdam, The Netherlands. Copyright1997 OPA (Overseas Publishers Association) Amsterdam B.V. ISBN90-5702-117-X, the entirety of which is hereby incorporated herein byreference.

Alternatively, compounds of the present invention may be prepared bysemi-synthetic processes starting from other compounds found in extractsof black cohosh and related cimicifuga species, whether from roots andrhizome or aerial parts of these plants. One of ordinary skill in theart will recognize that synthetic precursors may be obtained from one ormore cimicifuga species including, but not limited to, Cimicifugaracemosa, Cimicifuga dahurica, Cimicifuga foetida, Cimicifugaheracleifolia, Cimicifuga japonica, Cimicifuga acerina, Cimicifugaacerima, Cimicifuga simplex, and Cimicifuga elata, Cimicifugacalthaefolia, Cimicifuga frigida, Cimicifuga laciniata, Cimicifugamairei, Cimicifuga rubifolia, Cimicifuga americana, Cimicifugabiternata, and Cimicifuga bifida or a variety thereof. This may beaccomplished either by chemical or biological transformation of anisolated compound or an extract fraction or mixture of compounds.Chemical transformation may be accomplished by, but not limited to,manipulation of temperature, pH, and/or treatment with various solvents.Biological transformation may be accomplished by, but not limited to,treatment of an isolated compound or an extract fraction or mixture ofcompounds with plant tissue, plant tissue extracts, othermicrobiological organisms or an isolated enzyme from any organism.

In certain embodiments, the present invention provides an extract ofblack cohosh root wherein said extract comprises at least 10% by weightof a compound of the present invention. In other embodiments, thepresent invention provides an extract of black cohosh root wherein saidextract comprises from about 10% by weight to about 50% by weight of acompound of the present invention. In still other embodiments, thepresent invention provides an extract of black cohosh root wherein saidextract comprises from about 10% by weight to about 50% by weight of acompound of the present invention, wherein said extract is substantiallyfree of actein.

According to another embodiment, the present invention provides acompound of formula II, for use in methods of the present invention,substantially free of other compounds found in black cohosh root. Asused herein, the term “substantially free” means that the compound ismade up of a significantly greater proportion of a compound of formulaII as compared with the compound as found in black cohosh root orextracts thereof. In some embodiments, the present invention provides acompound of formula II in an amount of about 1 weight percent to about99 weight percent. In certain embodiments, the compound of formula II isprovided in greater than about 80% chemical purity. In otherembodiments, the compound of formula II is provided in greater thanabout 90% chemical purity. In other embodiments, the compound of formulaII contains no more than about 10.0 area percent HPLC of othercomponents of black cohosh root relative to the total area of the HPLCchromatogram. In other embodiments, the compound of formula II containsno more than about 8.0 area percent HPLC of other components of blackcohosh root relative to the total area of the HPLC chromatogram, and instill other embodiments, no more than about 3 area percent.

Methods to determine whether the compounds of the present invention arein a form substantially free of other compounds normally found in blackcohosh root are known to one of ordinary skill in the art as describedbelow. Compounds that were previously isolated, and identified, fromblack cohosh root include certain cycloartanol-based triterpenesincluding acteol, acetylacteol, 26-deoxyacteol, cimigenol, actein,26-deoxyactein, and cimicifugoside. (E)-Isoferulic acid and theisoflavone formononetin have also been isolated and identified.Representatives of these compounds have the following structures:

Accordingly, another embodiment of the present invention provides acompound of formula II, useful in methods of the present invention,substantially free of one or more of acteol, acetylacteol,26-deoxyacteol, cimigenol, actein, 26-deoxyactein, and cimicifugoside.In certain embodiments, the present invention provides a compound offormula II, for use in methods of the present invention, substantiallyfree of acteol, acetylacteol, 26-deoxyacteol, cimigenol, actein,26-deoxyactein, and cimicifugoside.

According to another embodiment, the present invention provides anextract of black cohosh root enriched in a compound of formula II,useful in methods of the present invention, with a diminished amount ofone or more of acteol, acetylacteol, 26-deoxyacteol, cimigenol, actein,26-deoxyactein, and cimicifugoside. According to yet another embodiment,the present invention provides an extract of black cohosh root enrichedin a compound of formula II, useful in methods of the present invention,with a diminished amount of each of acteol, acetylacteol,26-deoxyacteol, cimigenol, actein, 26-deoxyactein, and cimicifugoside.

A variety of techniques are well known in the art for extracting,isolating, and/or purifying individual active components of black cohoshroot. The present invention encompasses both the identification of suchactive components as described herein and the incorporation of suchcomponents into the compositions of the present invention as describedherein.

Individual active components of black cohosh extracts may be identifiedas described herein and may be isolated and/or purified using anytechniques known in the art. The active component may be purified fromthe root itself in any form or the decoction of a mixture of an extractof the present invention or a commercially available extract, amongothers. Various techniques that may be employed in the purificationinclude filtration, selective precipitation, extraction with organicsolvents, extraction with aqueous solvents, column chromatography(silica gel), high performance liquid chromatography (HPLC) and othermethods known to one of ordinary skill in the art.

According to certain embodiments, the present extracts useful in methodsof the present invention are those using an isolated fraction from blackcohosh root. An isolated fraction means a subsidiary amount of rootsubstances which has been removed, for example, by chromatographicmeans, distillation, precipitation, extraction, filtration or in otherways from the root itself. In other embodiments, the root extracts andfractions are removed therefrom by chromatography, distillation,precipitation, or extraction. Such extraction and isolation techniquesare well known to one of ordinary skill in the art. The details of someof these techniques are set forth in the Examples section below.

According to other embodiments of the present invention, the presenceand purity of the active compound is assessed by chemical methodsincluding nuclear magnetic resonance (NMR) spectroscopy, massspectrometry, infrared spectroscopy (IR), ultra-violet visiblespectroscopy, elemental analysis, and polarimetry, refractometry, toname but a few Such methods of analysis are known to one of ordinaryskill in the art. In other embodiments, the chemical structure of activecompounds isolated from black cohosh root is determined by methods knownto one of ordinary skill in the art, including NMR, mass spectrometry,infrared spectroscopy (IR), ultra-violet visible spectroscopy, elementalanalysis, polarimetry, refractometry, and X-ray crystallography, to namebut a few.

Although certain exemplary embodiments are described above and herein,it will be appreciated that the root extracts of the present inventioncan be prepared according to the methods described generally above usingappropriate starting materials by methods generally available to one ofordinary skill in the art.

5. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

According to another aspect of the present invention, pharmaceuticallyacceptable compositions are provided, wherein these compositionscomprise any of the compounds as described herein, and optionallycomprise a pharmaceutically acceptable carrier, adjuvant or vehicle. Incertain embodiments, these compositions optionally further comprise oneor more additional therapeutic agents. In certain embodiments, thepresent invention provides a composition comprising a compound offormula I, prepared by methods of the present invention, and apharmaceutically acceptable carrier, adjuvant, or vehicle.

It will also be appreciated that certain compounds of the presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable salt thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or a pharmaceutically active metabolite orresidue thereof. As used herein, the term “pharmaceutically activemetabolite or residue thereof” means that a metabolite or residuethereof is also a pharmaceutically active compound in accordance withthe present invention.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersable products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl sulfonate and aryl sulfonate.

The compositions of the present invention may additionally comprise apharmaceutically acceptable carrier, adjuvant, or vehicle, which, asused herein, includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutically acceptable compositionsand known techniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The compositions provided by the present invention can be employed incombination therapies, that is the present compositions can beadministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutic agents or medical procedures. The particularcombination of therapies (therapeutic agents or procedures) to employ ina combination regimen will take into account compatibility of thedesired therapeutic agents and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, a compound described herein may be administered concurrentlywith another therapeutic agent used to treat the same disorder), or theymay achieve different effects (e.g., control of any adverse effects).

For example, known agents useful for treating neurodegenerativedisorders may be combined with the compositions of this invention totreat neurodegenerative disorders, such as Alzheimer's disease. Examplesof such known agents useful for treating neurodegenerative disordersinclude, but are not limited to, treatments for Alzheimer's disease suchas acetylcholinesterase inhibitors, including donepezil, memantine (andrelated compounds as NMDA inhibitors), Exelon®; treatments forParkinson's disease such as L-DOPA/carbidopa, entacapone, ropinrole,pramipexole, bromocriptine, pergolide, trihexephendyl, and amantadine;agents for treating Multiple Sclerosis (MS) such as beta interferon(e.g., Avonex® and Rebif®), Copaxone®, and mitoxantrone; riluzole, andanti-Parkinsonian agents. For a more comprehensive discussion of updatedtherapies useful for treating neurodegenerative disorders, see, a listof the FDA approved drugs at http://www.fda.gov, and The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

In other embodiments, the compounds of the present invention arecombined with other agents useful for treating neurodegenerativedisorders, such as Alzheimer's disease, wherein such agents includebeta-secretase inhibitors, gamma-secretase inhibitors, aggregationinhibitors, farnesyl transferase inhibitors, metal chelators,antioxidants, and neuroprotectants.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a compound of formula I,prepared according to methods of the present invention, an additionaltherapeutic agent, and a pharmaceutically acceptable carrier, adjuvant,or vehicle.

Other examples of agents the inhibitors of this invention may also becombined with include, without limitation: treatments for asthma such asalbuterol and Singulair®; agents for treating schizophrenia such aszyprexa, risperdal, seroquel, and haloperidol; anti-inflammatory agentssuch as corticosteroids, TNF blockers, IL-1 RA, azathioprine,cyclophosphamide, and sulfasalazine; immunomodulatory andimmunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophosphamide,azathioprine, and sulfasalazine; neurotrophic factors such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, agents for treatingcardiovascular disease such as beta-blockers, ACE inhibitors, diuretics,nitrates, calcium channel blockers, and statins; agents for treatingliver disease such as corticosteroids, cholestyramine, interferons, andanti-viral agents; agents for treating blood disorders such ascorticosteroids, anti-leukemic agents, and growth factors; and agentsfor treating immunodeficiency disorders such as gamma globulin.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. In certain embodiments, the amount of additionaltherapeutic agent in the present compositions will range from about 50%to 100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

In an alternate embodiment, the methods of this invention that utilizecompositions that do not contain an additional therapeutic agent,comprise the additional step of separately administering to said patientan additional therapeutic agent. When these additional therapeuticagents are administered separately they may be administered to thepatient prior to, sequentially with or following administration of thecompositions of this invention.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the disorder being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

In some embodiments, the present invention provides a compositioncontaining a compound of formula I, prepared according to methods of thepresent invention, in an amount of about 1 weight percent to about 99weight percent. In other embodiments, the composition containing acompound of formula I, prepared according to methods of the presentinvention, contains no more than about 10.0 area percent HPLC of othercomponents of black cohosh root relative to the total area of the HPLCchromatogram. In other embodiments, the composition containing acompound of formula I, prepared according to methods of the presentinvention, contains no more than about 8.0 area percent HPLC of othercomponents of black cohosh root relative to the total area of the HPLCchromatogram, and in still other embodiments, no more than about 3 areapercent.

Uses of Compounds and Pharmaceutically Acceptable Compositions

The compounds prepared by methods of the present invention are usefulfor modulating and/or inhibiting amyloid-beta (1-42) peptide productionin a patient. Accordingly, the compounds of the present invention areuseful for treating, or lessening the severity of, disorders associatedwith amyloid-beta (1-42) peptide production in a patient.

The compounds, extracts, and compositions, according to the method ofthe present invention, may be administered using any amount and anyroute of administration effective for treating or lessening the severityof a neurodegenerative disorder. The exact amount required will varyfrom subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the infection, the particularagent, its mode of administration, and the like.

In certain embodiments, the present invention provides a method formodulating and/or inhibiting amyloid-beta (1-42) peptide production in apatient, wherein said method comprises administering to said patient acompound of formula I, prepared according to methods of the presentinvention, or a pharmaceutically acceptable composition comprising saidcompound. In other embodiments, the present invention provides a methodof selectively modulating and/or inhibiting amyloid-beta (1-42) peptideproduction in a patient, wherein said method comprises administering tosaid patient a compound of formula I, prepared according to methods ofthe present invention, or a pharmaceutically acceptable compositionthereof. In still other embodiments, the present invention provides amethod of reducing amyloid-beta (1-42) peptide levels in a patient,wherein said method comprises administering to said patient a compoundof formula I, prepared according to methods of the present invention, ora pharmaceutically acceptable composition thereof. In other embodiments,the present invention provides a method for reducing amyloid-beta (1-42)peptide levels in a cell, comprising contacting said cell with acompound of formula I, prepared according to methods of the presentinvention. Another embodiment provides a method for reducingamyloid-beta (1-42) in a cell without substantially reducingamyloid-beta (1-40) peptide levels in the cell, comprising contactingsaid cell with a compound of formula I, prepared according to methods ofthe present invention. Yet another embodiment provides a method forreducing amyloid-beta (1-42) in a cell and increasing at least one ofamyloid-beta (1-37) and amyloid-beta (1-39) in the cell, comprisingcontacting said cell with a compound of formula I, prepared according tomethods of the present invention.

As used herein, the term “reducing” or “reduce” refers to the relativedecrease in the amount of an amyloid-beta achieved by administering acompound of formula I, prepared according to methods of the presentinvention, as compared to the amount of that amyloid-beta in the absenceof administering a compound of formula I, prepared according to methodsof the present invention. By way of example, a reduction of amyloid-beta(1-42) means that the amount of amyloid-beta (1-42) in the presence of acompound of formula I, prepared according to methods of the presentinvention, is lower than the amount of amyloid-beta (1-42) in theabsence of a compound of formula I, prepared according to methods of thepresent invention.

In still other embodiments, the present invention provides a method forselectively reducing amyloid-beta (1-42) peptide levels in a patient,wherein said method comprises administering to said patient a compoundof formula I, prepared according to methods of the present invention, ora pharmaceutically acceptable composition thereof. In certainembodiments, the present invention provides a method for reducingamyloid-beta (1-42) peptide levels in a patient without substantiallyreducing amyloid-beta (1-40) peptide levels, wherein said methodcomprises administering to said patient a compound of formula I,prepared according to methods of the present invention, or apharmaceutically acceptable composition thereof.

In certain embodiments, the present invention provides a method forreducing amyloid-beta (1-42) peptide levels in a patient and increasingat least one of amyloid-beta (1-37) and amyloid-beta (1-39), whereinsaid method comprises administering to said patient a compound offormula I, prepared according to methods of the present invention, or apharmaceutically acceptable composition thereof.

The term “increasing” or “increase,” as used herein in reference to anamount of an amyloid-beta, refers to the relative rise in the amount ofan amyloid-beta achieved by administering a compound of formula I,prepared according to methods of the present invention, (or contacting acell with a compound of formula I) as compared to the amount of thatamyloid-beta in the absence of administering a compound of formula I,prepared according to methods of the present invention, (or contacting acell with a compound of formula I). By way of example, an increase ofamyloid-beta (1-37) means that the amount of amyloid-beta (1-37) in thepresence of a compound of formula I, prepared according to methods ofthe present invention, is higher than the amount of amyloid-beta (1-37)in the absence of a compound of formula I, prepared according to methodsof the present invention. For instance, the relative amounts of eitherof amyloid-beta (1-37) and amyloid-beta (1-39) can be increased eitherby an increased production of either of amyloid-beta (1-37) andamyloid-beta (1-39) or by a decreased production of longer amyloid-betapeptides, e.g., amyloid-beta (1-40) and/or amyloid-beta (1-42). Inaddition, it will be appreciated that the term “increasing” or“increase,” as used herein in reference to an amount of an amyloid-beta,also refers to the absolute rise in the amount of an amyloid-betaachieved by administering a compound of formula I, prepared according tomethods of the present invention. Thus, in certain embodiments, thepresent invention provides a method for increasing the absolute level ofat least one of amyloid-beta (1-37) and amyloid-beta (1-39), whereinsaid method comprises administering to said patient a compound offormula I, prepared according to methods of the present invention, or apharmaceutically acceptable composition thereof. In other embodiments,the present invention provides a method for increasing the level of atleast one of amyloid-beta (1-37) and amyloid-beta (1-39), wherein theincrease is relative to the amount of longer amyloid-beta peptides,e.g., amyloid-beta (1-40) and/or amyloid-beta (1-42), or totalamyloid-beta, wherein said method comprises administering to saidpatient a compound of formula I, prepared according to methods of thepresent invention, or a pharmaceutically acceptable composition thereof.

One of ordinary skill in the art will appreciate that overall ratio ofamyloid-beta peptides is significant where selective reduction ofamyloid-beta (1-42) is especially advantageous. In certain embodiments,the present compounds reduce the overall ratio of amyloid-beta (1-42)peptide to amyloid-beta (1-40) peptide. Accordingly, another aspect ofthe present invention provides a method for reducing the ratio ofamyloid-beta (1-42) peptide to amyloid-beta (1-40) peptide in a patient,comprising administering to said patient a compound of formula I,prepared according to methods of the present invention, or apharmaceutically acceptable composition thereof. In certain embodiments,the ratio of amyloid-beta (1-42) peptide to amyloid-beta (1-40) peptideis reduced from a range of about 0.1 to about 0.4 to a range of about0.05 to about 0.08.

In other embodiments, the present invention provides a method forreducing the ratio of amyloid-beta (1-42) peptide to amyloid-beta (1-40)peptide in a cell, comprising contacting the cell with a compound offormula I, prepared according to methods of the present invention. Incertain embodiments, the ratio of amyloid-beta (1-42) peptide toamyloid-beta (1-40) peptide is reduced from a range of about 0.1 toabout 0.4 to a range of about 0.05 to about 0.08.

According to one aspect, the present invention provides a method fortreating or lessening the severity of a disorder associated withamyloid-beta (1-42) peptide, wherein said method comprises administeringto said patient a compound of formula I, or a pharmaceuticallyacceptable composition thereof. Such disorders include neurodegenerativedisorders such as Alzheimer's disease (familial and sporadic),Parkinson's disease, and Down's syndrome.

Such disorders also include inclusion body myositis (deposition ofA-beta in peripheral muscle, resulting in peripheral neuropathy),cerebral amyloid angiopathy (amyloid in the blood vessels in the brain),and mild cognitive impairment.

“High A-beta42” is a measurable condition that precedes symptomaticdisease, especially in familial patients, based on plasma, CSFmeasurements, and/or genetic screening. This concept is analogous to therelationship between elevated cholesterol and heart disease. Thus,another aspect of the present invention provides a method for preventinga disorder associated with elevated amyloid-beta (1-42) peptide, whereinsaid method comprises administering to said patient a compound offormula I, prepared by methods of the present invention, or apharmaceutically acceptable composition thereof.

In other embodiments, the present invention provides a method fortreating a diseases where A-beta amyloidosis may be an underlying aspector a co-existing and exacerbating factor, wherein said method comprisesadministering to said patient a compound of formula I, prepared bymethods of the present invention, or a pharmaceutically acceptablecomposition thereof.

In still other embodiments, the present invention provides a method fortreating a disorder in a patient, wherein said method comprisesadministering to said patient a compound of formula I, or apharmaceutically acceptable composition thereof, and wherein saiddisorder is Lewy body dementia (associated with deposition ofalpha-synuclein into Lewy bodies in cognitive neurons; a-synuclein ismore commonly associated with deposits in motor neurons and the etiologyof Parkinson's disease), Parkinson's disease, cataract (where a-beta isaggregating in the eye lens), Tauopathies (e.g. frontotemporaldementia), Huntington's disease, ALS/Lou Gerhig's disease, Type 2diabetes (IAPP aggregates in pancreatic islets, is similar in size andsequence to A-beta and having type 2 diabetes increases risk ofdementia), Transthyretin amyloid disease (TTR, an example of thisdisease is in heart muscle contributing to cardiomyopathy), priondisease, and Creutzfeldt-Jakob disease (CJD).

In other embodiments, the present invention provides a method fortreating or lessening the severity of Alzheimer's disease in a patient,wherein said method comprises administering to said patient a compoundof formula I, prepared by methods of the present invention, or apharmaceutically acceptable composition thereof.

Without wishing to be bound by any particular theory, it is believedthat the present compounds are modulators of gamma-secretase whichselectively reduce levels of amyloid-beta (1-42). Accordingly, anotherembodiment of the present invention provides a method of modulatinggamma-secretase in a patient, comprising administering to said patient acompound of formula I, prepared according to methods of the presentinvention, or pharmaceutically acceptable composition thereof. Incertain embodiments, the present compounds are inhibitors ofgamma-secretase. Said method is useful for treating or lessening theseverity of any disorder associated with gamma-secretase. Such disordersinclude, without limitation, neurodegenerative disorders, e.g.Alzheimer's disease.

The Notch/Delta signaling pathway is highly conserved across species andis widely used during both vertebrate and invertebrate development toregulate cell fate in the developing embryo. See Gaiano and Fishell,“The Role of Notch in Promoting Glial and Neural Stem Cell Fates” Annu.Rev. Neurosci. 2002, 25:471-90. Notch interacts with the gamma-secretasecomplex and has interactions with a variety of other proteins andsignaling pathways. Notchl competes with the amyloid precursor proteinfor gamma-secretase and activation of the Notch signaling pathwaydown-regulates PS-1 gene expression. See Lleo et al., “Notchl Competeswith the Amyloid Precursor Protein for γ-Secretase and Down-regulatesPresenilin-1 Gene Expression” Journal of Biological Chemistry 2003,48:47370-47375. Notch receptors are processed by gamma-secretase actingin synergy with T cell receptor signaling and thereby sustain peripheralT cell activation. Notch1 can directly regulate Tbx21 through complexesformed on the Tbx21 promoter. See Minter et al., “Inhibitors ofγ-secretase block in vivo and in vitro T helper type 1 polarization bypreventing Notch upregulation of Tbx21,” Nature Immunology 2005,7:680-688. In vitro, gamma-secretase inhibitors extinguished expressionof Notch, interferon-gamma and Tbx21 in TH1-polarized CD4+ cells. Invivo, administration of gamma-secretase inhibitors substantially impededTH1-mediated disease progression in the mouse experimental autoimmuneencephalomyelitis model of multiple sclerosis suggesting the possibilityof using such compounds to treat TH1-mediated autoimmunity See Id.Inhibition of gamma-secretase can alter lymphopoiesis and intestinalcell differentiation (Wong et al., “Chronic Treatment with theγ-Secretase Inhibitor LY-411,575 Inhibits β-Amyloid Peptide Productionand Alters Lymphopoiesis and Intestinal Cell Differentiation” Journal ofBiological Chemistry 2004, 26:12876-12882), including the induction ofgoblet cell metaplasia. See Milano et al., “Modulation of NotchProcessing by g-Secretase Inhibitors Causes Intestinal Goblet CellMetaplasia and Induction of Genes Known to Specify Gut Secretory LineageDifferentiation” Toxicological Sciences 2004, 82:341-358.

Strategies that can alter amyloid precursor protein (“APP”) processingand reduce the production of pathogenic forms of amyloid-beta withoutaffecting Notch processing are highly desirable. Moreover, as describedabove, the inhibition of gamma-secretase has been shown in vitro and invivo to inhibit the polarization of Th cells and is therefore useful fortreating disorders associated with Th1 cells. Th1 cells are involved inthe pathogenesis of a variety of organ-specific autoimmune disorders,Crohn's disease, Helicobacter pylori-induced peptic ulcer, acute kidneyallograft rejection, and unexplained recurrent abortions, to name a few.

According to one embodiment, the invention relates to a method ofinhibiting the formation of Th1 cells in a patient comprising the stepof administering to said patient a compound of the present invention, ora composition comprising said compound. In certain embodiments, thepresent invention provides a method for treating one or more autoimmunedisorders, including irritable bowel disorder, Crohn's disease,rheumatoid arthritis, psoriasis, Helicobacter pylori-induced pepticulcer, acute kidney allograft rejection, multiple sclerosis, or systemiclupus erythematosus, wherein said method comprises administering to saidpatient a compound of formula I, prepared according to methods of thepresent invention, or a pharmaceutically acceptable compositioncomprising said compound.

In certain embodiments, the present invention provides a method formodulating and/or inhibiting amyloid-beta peptide production, withoutaffecting Notch processing, in a patient, wherein said method comprisesadministering to said patient a compound of formula I, preparedaccording to methods of the present invention, or a pharmaceuticallyacceptable composition comprising said compound.

In certain embodiments, the present invention provides a method forinhibiting amyloid-beta (1-42) peptide production, without affectingNotch processing, in a patient, wherein said method comprisesadministering to said patient a compound of formula I, preparedaccording to methods of the present invention, or a pharmaceuticallyacceptable composition comprising said compound.

In certain embodiments, the present invention provides a method forreducing amyloid-beta (1-42) peptide levels in a patient and increasingat least one of amyloid-beta (1-37) and amyloid-beta (1-39), withoutaffecting Notch processing, wherein said method comprises administeringto said patient a compound of formula I, prepared according to methodsof the present invention, or a pharmaceutically acceptable compositionthereof.

Accordingly, another aspect of the present invention provides a methodfor reducing the ratio of amyloid-beta (1-42) peptide to amyloid-beta(1-40) peptide in a patient, without affecting Notch processing,comprising administering to said patient a compound of formula I,prepared according to methods of the present invention, or apharmaceutically acceptable composition thereof. In certain embodiments,the ratio of amyloid-beta (1-42) peptide to amyloid-beta (1-40) peptideis reduced from a range of about 0.1 to about 0.4 to a range of about0.05 to about 0.08.

The compounds of the invention are preferably formulated in dosage unitform for ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof agent appropriate for the patient to be treated. It will beunderstood, however, that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specificeffective dose level for any particular patient or organism will dependupon a variety of factors including the disorder being treated and theseverity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed, and like factors wellknown in the medical arts. The term “patient,” as used herein, means ananimal, preferably a mammal, and most preferably a human.

EXAMPLES

The following experimentals describe the isolation and/or enrichment ofcompounds for use in methods of the present invention. The black cohoshextract, utilized in the separation protocol described below, wasobtained as a custom order from Boehringer Ingelheim Nutriceuticals.This extract is substantially equivalent to the USP preparation of blackcohosh extract, in which about 50% aqueous ethanol is used to extractpowdered root and then concentrated to near dryness.

As used herein, the compound numbers recited below correspond to thefollowing compounds:

Compound 1: β-D-Xylopyranoside,(3,12,16,23R,24R,25S,26S)-12-(acetyloxy)-16, 23:23,26:24,25-triepoxy-26-hydroxy-9,19-cyclolanostan-3-yl.

Also known as “actein.” C₃₇H₅₆O₁₁; Mol. Wt.: 676.83; Registry18642-44-9.

Compound 2: Cimigenol 3-β-D-xylopyranoside; C₃₅H₅₆O₉, Mol. Wt.: 620.81;Registry 27994-11-2.

Compound 3: Cimigenol 3-α-L-arabinoside. C₃₅H₅₆O₉, Mol. Wt.: 620.81;Registry 256925-92-5.

Compound 4: 24-O-Acetylhydroshengmanol 3-β-D-xylopyranoside. C₃₇H₆₀O₁₁,Mol. Wt.: 680.87; Registry 78213-32-8.

Compound 5: 24-O-Acetylhydroshengmanol 3-α-L-arabinopyranoside.C₃₇H₆₀O₁₁, Mol. Wt.: 680.87.

Compound 6: 24-O-Acetylhydroshengmanol 3-β-D-xylopyranoside(delta-16,17)-enol ether. C₃₇H₅₈O₁₀, Mol. Wt.: 662.85.

Compound 7: 24-O-Acetylhydroshengmanol 3-α-L-arabinopyranoside(delta-16,17)-enol ether. C₃₇H₅₈O₁₀, Mol. Wt.: 662.85.

Compound 8: 24-epi-24-O-Acetylhydroshengmanol 3-β-D-xylopyranoside.C₃₇H₆₀O₁₁, Mol. Wt.: 680.87.

Compound 9: 24-epi-24-O-Acetylhydroshengmanol 3-β-D-xylopyranoside(delta-16,17)-enol ether. C₃₇H₅₈O₁₀, Mol. Wt.: 662.85.

Isolation Protocol 1 Flash Column Chromatography

Black cohosh extract (15.6 g) was suspended in 150 ml of a 4-to-1 (v/v)methanol-water mixture at 25° C. Using a mechanical stirrer, theresulting slurry was vigorously stirred for 30 minutes at thistemperature, which resulted in a brown emulsion. To this emulsion, 51 gof silica gel (ICN silica 32-63 60 Å) was added with continued stirring.The mixture was concentrated at 25° C. in vacuo using a rotaryevaporator, until a largely homogenous beige-brown powder was obtained.This material was subjected to column chromatography on silica gel (ICNsilica 32-63 60 Å) using a 60 cm long glass column with 50 mm innerdiameter.

In preparation for the column chromatography silica gel was poured into500 ml of a 20-to-1 dichloromethane-methanol mixture, and the resultingslurry was poured into the glass column. The silica gel was allowed tosettle for 30 minutes, and covered with a 1 cm thick layer of sand.Subsequently, the extract absorbed onto silica was poured into 20-to-1dichloromethane-methanol mixture, and the resulting slurry was pouredonto the sand layer on top of the column. The silica column was theneluted with the following solvent mixtures under a pressure of 0.4 bar(argon):

1.0 ml of dichloromethane-methanol 20-to-1, followed by

770 ml of dichloromethane-methanol 10- to 1, followed by

800 ml of dichloromethane-methanol 7-to-1, followed by

550 ml of dichloromethane-methanol 5-to-1.

Eight 200 ml-fractions (labeled as sat14-0 through sat14-7) werecollected, followed by eleven 100-ml fractions (labeled as sat14-8through sat14-18). All fractions were analyzed by thin-layerchromatography (TLC), using Bakerflex silica plates, eluted with a5-to-1 dichloromethane-methanol solvent mixture. After development, thesilica gel plates were stained with anisaldehyde stain. Based on theresults of the TLC analyses, fractions sat14-9 through sat14-12 wereevaporated to dryness in vacuo at 25° C., and 10-mg samples of thesefractions were analyzed by ¹H-NMR spectroscopy, using CD₃OD as solvent.Spectra were analyzed with regard to the presence of a broad multipletat 2.53 ppm, and a 2.2-Hz doublet at 4.86 ppm, because these signals arecharacteristic for compounds 7 and 6. Additional dqf-COSY spectra ofthese four samples confirmed that the signals at 2.53 and 4.86 ppm infact belong to compounds 7 and 6. From the ¹H-NMR spectra of fractionsat14-10 it was concluded that this sample contained the highestconcentration of compounds 7 and 6, while slightly smaller amounts ofthese compounds could be detected in fraction sat14-9. Fraction sat14-11appeared to contain traces of 7 and 6, whereas these compounds could notbe detected in fraction sat14-12. Based on these results, fractionsat14-10 was chosen for further purification via HPLC. Alternatively,fraction sat14-9 could be used, to obtain additional amounts ofcompounds 4 through 7 as needed.

The major component of fraction sat14-10 was actein (1) (JNP 2002, 65,601-605), which crystallized from a methanolic solution of thisfraction. Pure actein was obtained through recrystallization. Majorcomponents of fraction sat14-11 were cimigenol beta-D-xylopyranoside (2)and cimigenol alpha-L-arabinoside (3), which crystallized from thisfraction as a mixture of roughly 2:1 (JNP 2000, 65, 905-910 and1391-1397).

Reversed-Phase HPLC Fractionation on C-18 Column

Fraction sat14-10 was dissolved in 3.5 ml of methanol. This solution wasfractionated by HPLC using a SUPELCO Discovery RP-18 column (25 cmlength, 10 mm inner diameter), and an AGILENT 1100 series HPLC system,including auto-injector and a diode array detector used for detection ofwavelength from 190-400 nm. A solvent gradient was employed, startingwith 30% (v/v) water in methanol for the first two minutes, followed bya linear decrease of water content reaching 100% methanol at 20 minutes.After 2 minutes at 100% methanol, water content was increased to 30% andmaintained at that concentration for another 8 minutes. For separationof the entire sample sat14-10, 100 injections of 35 μl each wererequired. Nine fractions were collected, which were labeled sat15-1through sat15-9. Compounds 4 through 7 were eluted in fractions sat15-1,15-2, 15-4 and15-5:

Reversed-Phase HPLC Fractionation on C-8 Column for the Isolation of 6,4, and 9

Fraction sat15-5 was dissolved in 1.5 ml of methanol. This solution wasfractionated by HPLC using a SUPELCO supelcosil LC-8 column (25 cmlength, 10 mm inner diameter), and the AGILENT 1100 series HPLC systemdescribed above. A solvent gradient was employed, starting with 40%(v/v) water in methanol for the first two minutes, followed by a lineardecrease of water content reaching 100% methanol at 20 minutes. After 2min at 100% methanol, water content was increased to 40% and maintainedat that concentration for another 8 minutes. For separation of theentire sample sat15-5, 50 injections of 30 μl each were required. Fivefractions were collected, which were labeled sat16-1 through sat16-5.Compound 6 was eluted in fraction sat16-3, whereas compound 4 eluted infraction sat16-1. A small amount of pure 9 was obtained in fractionsat15-5.

Reversed-Phase HPLC Fractionation on C-8 Column for the Isolation of 8

Fraction sat15-8 was dissolved in 0.65 ml of methanol. This solution wasfractionated by HPLC using a SUPELCO supelcosil LC-8 column (25 cmlength, 10 mm inner diameter), and the AGILENT 1100 series HPLC systemdescribed above. A solvent gradient was employed, starting with 40%(v/v) water in methanol for the first two minutes, followed by a lineardecrease of water content reaching 100% methanol at 20 minutes. After 2minutes at 100% methanol, water content was increased to 40% andmaintained at that concentration for another 8 minutes. Seven fractionswere collected, which were labeled sat18-1 through sat18-7. Compound 8was eluted in fraction sat18-6. NMR-spectroscopic analyses includingNOESY spectra showed that in methanolic solution compound 8interconverts with the corresponding ketone. Dilute methanolic solutionscontain about 4% ketone and 96% of the hemiacetal form.

Reversed-Phase HPLC Fractionation on C-8 Column for the Isolation of 7and 5

Fraction sat15-2 was dissolved in 0.5 ml of methanol. This solution wasfractionated by HPLC using a SUPELCO supelcosil LC-8 column (25 cmlength, 10 mm inner diameter), and the AGILENT 1100 series HPLC systemdescribed above. A solvent gradient was employed, starting with 40%(v/v) water in methanol for the first two minutes, followed by a lineardecrease of water content reaching 100% methanol at 20 minutes. After 2minutes at 100% methanol, water content was increased to 40% andmaintained at that concentration for another 8 minutes. Five fractionswere collected, which were labeled sat19-3 through sat19-7. Purecompound 7 was obtained in fraction sat19-7, whereas pure compound 5 wasobtained in fraction sat19-5.

Isolation Protocol 2

An alternative isolation/purification protocol is set forth below forisolating compound 6. One of ordinary skill in the art will recognizethat while isolating compound 6, other compounds of the presentinvention are enriched and/or isolated by this process.

This purification protocol utilized the following equipment:

-   -   (a) Hitachi HPLC system with diode array detector (DAD)    -   (b) Nova Prep™ 8000 SEMI-Preparative HPLC with Remote PC        Controller using LC ReSponder™ Application Software    -   (c) Hitachi UV Detector L-7400    -   (d) Sedex 55 evaporative light scattering (ELSD) detector    -   (e) 75L Biotage silica column (KP-Sil; P/N FKO-1107-19073; Lot        027075L)    -   (f) 75L Biotage C18 column(Bakerbond, 40μ)    -   (g) 75S Biotage C18 column(Vydac, 40μ)    -   (h) Analytical HPLC column: Phenomenex Luna C18, 3μ, 4.6×100 mm    -   (i) Semi-Preparative HPLC column: Phenomenex Luna C8 HPLC        column, 20×250 mm    -   (j) Semi-Preparative HPLC column: YMC AQ C18 HPLC column,        21.2×250 mm; and    -   (k) Preparative HPLC column: ES Industries C18 Preparative HPLC        column; 5×25 cm.

The analytical method utilized to determine the purity of compound 6 isas follows:

-   Column: Phenomenex Luna C18, 3μ, 4.6×100 mm-   Mobile Phase Isocratic elution with A. 35% Acetonitrile; B. 30%    Nanopure water containing 0.05% Acetic Acid; and C. 35% MeOH-   Flow Rate: 1 mL/min-   Detection: 205, 230 nm, DAD; and ELSD-   Run Time: 8 min-   Column Temperature: 32° C.    This method was used for the analysis of the extract, fractions, and    the final product. Compound 6 elutes at about 5.5 minutes under    these conditions.

50 g of crude black cohosh extract (“BCE”) was fractionated on a BiotageSilica cartridge (7.5×30 cm). After loading, the cartridge was elutedwith 5% MeOH/DCM (10 L) and 10% MeOH/DCM (5 L) and 500 ml fractions werecollected. The flow rate was 150-200 ml/min. The HPLC (UV at 230 nm)revealed the compound 6 was present in fractions 23 (2.6 g) and 24 (2.3g). The fraction 23 (F23) was selected for further purification on asemi-prep C8 column.

Ten runs were performed to get approximately 10 mg of compound 6. 50 mgof F23 in 0.3 ml of MeOH was loaded onto a Phenomenex Luna C-8 (21.2×250mm, 10p., 100 A) semi-prep column. The column was eluted at a flow rateof 9.9 mL/min with 70% MeOH in H₂O with UV monitoring at 205 nm. Thepeaks eluting at 35 min and 38 min as shown in semi-prep HPLC trace wereseparately collected.

The fractions collected for the peak at 35 min from the 10 runs werepooled and solvents evaporated at ambient temperature. The resultingsolids were dried on a lyophilizer to yield 10.3 mg of compound 6(2609-165-7). The HPLC of the product 2609-165-7 revealed a polarimpurity peak (11.3%) with retention time (RT) at 4.5 min, although theHPLC of individual fractions showed only one major peak. Apparentlycompound 6 converted slowly during the process to a more polar compound.It was found that the more polar compound was the deacetyl derivative ofcompound 6 as evident from SSI-MS which showed an intense [M+Na]⁺ peakat m/z 643 and proton NMR of the isolated impurity at 4.5 min in whichthe singlet for the acetyl methyl was absent.

A few stability experiments with compound 6 indicated that deacetylationoccurred in MeOH solution which is slightly basic. However, it wasstable in slightly acidic solution. Therefore, 2609-165-7 wasre-processed on the Luna C8 column using 70% MeOH/30% water containing0.05% AcOH as eluent to give 3.4 mg of compound 6 (2609-172-11).

In another process, 250 g of black cohosh extract (BCE) was stirred with1250 mL of MeOH for 1 hr at room temperature in a beaker. Not all thesolids dissolved but HPLC analysis of a filtrate indicated that allcompound 6 in the starting extract dissolved (˜250 mg). Nonetheless theunfiltered mixture was added to 750 g of silica gel (ICN, 60-200μ) in a5 L round bottom flask. The MeOH was removed on the rotovap with the aidof vacuum to a dry powder weighing 1100 g with 9% residual MeOH.

The BCE dried on silica preparation was divided into four parts ofapproximately 270 g each. The mixture was loaded into the SIM (sampleinjection module) and first washed with 500-600 mL of methylene chlorideto remove non-polars and residual MeOH. The SIM was connected to the 75Lsilica column (KP-Sil; P/N FKO-1107-19073; Lot 027075L; 7.5×25 cm or1750 mL). The main column was radial compressed at 60 psi. The systemwas eluted with acetone at a flow rate of amount 100 mL/min, and500-1000 mL fractions were collected. After the elution of compound 6the column was washed with 1.0 L of MeOH and re-equilibrated with 2 L ofacetone. Compound 6 was observed to elute primarily in Fraction 3 (1000mL) after approximately 900-1000 mL of acetone had eluted from thecolumn in Fractions 1 and 2. The first four runs yielded approximately224 mg of compound 6. A second batch of starting material for the silicaBiotage was prepared from 100 g of BCE and 500 mL of MeOH and 300 g ofsilica. Two additional Biotage runs (5 and 6) were done similar to thefirst four with this starting material yielding another 93 mg ofcompound 6. The product pools from the six runs were combined andevaporated to a dry solid under reduced pressure.

The dried solids (90 g) from the silica Biotage were dissolved in 720 mLof MeOH and 480 mL of H₂O was added slowly with stirring. Some darktar-like solids precipitated out and were removed on a filter. Thecloudy filtrate was loaded on a 75L (7.5×25 cm) Bakerbond 60 Å, 40μBiotage C18 column. After the loading, which tested negative fromcompound 6, the column was washed with 5 L of 60% (v/v) MeOH/H₂Ofollowed by 4 L of 70% MeOH/H₂O, and then eluted compound 6 using 4 L of80% MeOH/H₂O. After the elution the column was washed with 2 L of MeOH.The flow rate was about 60 mL/min throughout and the MeOH/H₂O mobilephases contained 0.05% acetic acid in order to prevent degradation ofcompound 6. The product pool (4 L) was concentrated under reducedpressure until essentially all the MeOH was removed and the resultingprecipitated solids collected on a Buchner funnel and dried with the aidof high vacuum at room temperature.

The tar-like solids removed via filtration from the first large-scaleC18 feed preparation and containing about 32 mg of compound 6 weredissolved in 2 L of MeOH wash from the large-scale experiment and whichcontained about 22 mg of Compound 6. The mixture was evaporated to 1 Land mixed with 0.67 L of water. Some tar-like solids precipitated outwhich were collected on a filter, dissolved in 200 mL of MeOH, and mixedwith 134 mL of water. This mixture was also filtered to remove a smallamount of tar and the filtrate combined with the first filtrate andloaded on a 75S (7.5×9.0 cm; 400 mL) Vydac 300 Å, 40μ Biotage C18column. The column was washed with 1 L of 60% MeOH/H₂O and 2 L of 70%MeOH/H₂O, and eluted with 1 L of 80% MeOH/H₂O (mobile phases alsocontained 0.05% acetic acid). The product pool was evaporated and thesolids collected by filtration similar to the large-scale Biotageexperiment.

The first product pool (16.69 g) from the C18 Biotage column was mixedwith 70 mL of MeOH. The mixture was sonicated, and the precipitate wasremoved by filtration. The filtrate was chromatographed (five runs, 14mL each) on an ES Industries Chromegabond WR C18 column at flow rate of177 mL/min using 70% MeOH/30% water containing 0.05% AcOH as eluent. Thefractions from the 6-14 minutes of each run were combined, andevaporated to remove MeOH. The precipitate after removal of MeOH wascollected by centrifugation, and dried on a lyophilizer to give 6.6 gdried solids 2609-173-16 (compound 6, 3.2%).

The second product pool (4.3 g) from the C18 Biotage column wasprocessed in similar manner to give 2.0 g dried solids 2609-173-27(compound 6, 3.06%). 2609-173-16 and 2609-176-27 were combined to yield8.6 g of 2609-174-6.

2609-174-6 (400 mg) was dissolved in 1.3 mL of MeOH containing 0.1%AcOH. The solution was loaded onto a Phenomenex Luna C8 column which waseluted at flow rate of 24 mL/min with 68% MeOH/32% water containing0.05% AcOH.

Based on analytical HPLC, the fractions from the 15.8 to 19.8 minute ofeach run (total 22 runs) were combined, evaporated to remove MeOH, andlyophilized to dryness to give 2609-174-28 (1.4 g containing 12.6% ofcompound 6). 2609-174-28 was used for the final isolation of compound 6on a YMC-AQ C18 column. A total of 28 runs were performed.

2609-174-28 (50 mg) was dissolved in 0.25 mL of MeOH containing 0.1%AcOH. The solution was injected into the YMC AQ C18 column. The columnwas eluted at 9.9 mL/min with 70% MeOH/30% water containing 0.05% AcOH.Based on analytical HPLC profiles, selected fractions, typically between48.4-50.4 minute, from the 28 runs were pooled, evaporated, andlyophilized to yield compound 6 (2609-176-30, 85 mg).

The fractions that were collected immediately before 48.4 min andcontained mainly compound 6 were also combined, and dried to give2609-176-35 (50 mg). 2609-176-35 was re-processed (3 runs) using thesame column and mobile phase to yield another lot of compound 6 whichwas combined with 2609-176-30 to give 102 mg of product (2609-177-10)with ˜95% chromatography purity.

Dehydration Reaction

Using the conversion of compound 4 to compound 6 to exemplify, thefollowing procedures describe the conversion of a compound of formula IIto a compound of formula I.

The conversion of compound 4 to compound 6 was monitored by thefollowing HPLC methods:

Method 1: A 4.6×100 mm, 3μ Luna(2) C18 column from Phenomenex (P/N00D-4251-E0) was used with a mobile phase of 35/30/35 MeCN/H₂O/MeOH(isocratic) for 8 min. Detection was via ELSD and UV at 205 and 230 nm.

Method 2: A 4.6×100 mm, 3μ Luna(2) C18 column from Phenomenex (P/N00D-4251-E0) was used with a mobile phase of 75/25 MeOH/H₂O (Method 2)at 1 mL/min. Detection was via ELSD and UV at 205 and 230 nm. The Method2 was used primarily for the black cohosh extract (BCE) experiments.

Conversion of Purified Compound 4 to Compound 6 with TFA: 1. 9 mg ofpurified compound 4 (2631-61-9; 92% purity) was sonicated with 2 mL ofMeOH (w/0.1% HOAc) then treated with 0.040 mL of TFA (4%) and after 1 hrthe reaction was quenched with 6 N NH₄OH to pH 6.

A second conversion experiment was done by dissolving 10 mg of compound4 (2631-61-15; 83% purity) in 3 mL of MeOH and adding 0.15 mL (5%) TFA.After sitting at room temperature for 40 min, the reaction mixture wasquenched with 6 N NH₄OH to pH 6. The HPLC analysis (ELSD) indicated thatmost of compound 4 had converted to compound 6. The HPLC analysis(Method 1) of the starting material indicated compound 4 was 2.7 mg/mlbefore the reaction and 0.5 mg/ml after and that compound 6 was 0.84mg/ml after the conversion.

Conversion of Crude Compound 4 to Compound 6:

Step A: Preparation of Methanolic BCE:

1.00 g of black cohosh extract (BCE) was sonicated at room temperaturefor 1 hr with 15.0 mL of methanol containing 0.1% acetic acid (HOAc).The mixture was filtered through a 0.45 micron PTFE membrane filter intoa vial.

Step B(1): Acid-Treatment of Methanol BCE:

(Experiment 1) 1.0 mL of the methanolic BCE was treated with 0.05 mL ofTFA in an HPLC vial. The untreated control and the treated sample wereanalyzed by HPLC after 0.5 hr, 2.5 hr, 3.5 hr, and 2.5 days. Treatmentof the methanolic extract of BCE with 5% TFA appeared to completelyconvert compound 4 to compound 6 in about 3.5 hr.

Step B(2): Acid-Treatment of Methanol BCE:

(Experiment 2) Fresh BCE (1 g in 15 mL of MeOH) was used in a secondseries of experiments in which compound 4 and compound 6 werequantitated against pure compound 4 and compound 6 standards. 1.0 mL ofthe BCE methanolic solution was treated with 30, 50, and 100 μL of TFAin a HPLC vial and the samples analyzed over a period of 6 hr by theHPLC Method 1. Compound 6 was quantitated at 230 nm and Peak 2 wasquantitated using both ELSD and UV at 205 nm. Analysis of the untreatedcontrol indicated that compound 6 in the BCE was 0.135%, while compound4 was a major component of the extract and was found to be 2.49% usingELSD and 1.95% using UV 205 nm. The data indicated that the cleanestconversion with the highest recovery of compound 6 took place in thesample treated with 3% TFA for a period of 2-2.5 hr. In this samplecompound 6's concentration in the acid-treated extract increased from0.090 mg/mL to 0.64 mg/mL (7.1× or 611%) while the assay for compound 4indicated that it decreased from 1.30 mg/mL to 0.2 mg/mL (85% loss usingUV₂₀₅). The two experiments indicated that the acid treatment increasedthe amount of Compound 3 in the methanolic BCE by 7-8 fold.

Biological Assays A. Assay to Determine the Ability of a Compound of thePresent Invention to Inhibit Aβ-42

Compounds of the present invention, and extracts comprising saidcompounds, may be assayed as inhibitors of amyloid-beta (1-42) peptidein vitro or in vivo. Such assay methods are described in detail in U.S.Pat. No. 6,649,196, the entirety of which is hereby incorporated hereinby reference.

Compounds of the present invention were found to selectively loweramyloid-beta (1-42) peptide according to the cell-based assay performedin substantially the same manner as described in U.S. Pat. No.6,649,196.

B. Assay to Determine Ability of a Compound of Formula I to Affect theRatio of Total Aβ

Compounds of the present invention were assayed to determine theireffect on the total ration of amyloid-β (1-42) peptide in vitro using anassay protocol substantially similar to that described by Wang et al.,J. Biol. Chem. 1996, 50:31894-31902, The Profile of Soluble Amyloid βProtein in Cultured Cell Media, the entirety of which is herebyincorporated herein by reference. This assay quantifies amyloid-βprotein using immunoprecipitation and mass spectrometry (IP-MS). Usingcompound 6 to exemplify, it was found that this compound reducedamyloid-β (1-42) peptide, while increasing amyloid-β (1-37) peptide andamyloid-β (1-39) peptide.

Compound 6 was also assayed according to the method described in Wang etal., in 7W cells (APP_(wt)) and 7PA2 cells (APP_(V717F)). The APP₇₁₇mutations increase the relative amount of amyloid-β (1-42) peptide. Inthis assay, it was shown that compound 6 reduces amyloid-β (1-42)peptide while increasing amyloid-β (1-39) peptide.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

1. A method for preparing a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: each of Ring A,Ring B, Ring C, Ring D, and Ring E is independently saturated, partiallyunsaturated or aromatic; G is S, CH₂, NR, or O; R¹ and R² are eachindependently halogen, R, OR, a suitably protected hydroxyl group, SR, asuitably protected thiol group, N(R)₂, or a suitably protected aminogroup, or R¹ and R² are taken together to form a 3-7 membered saturated,partially unsaturated, or aryl ring having 0-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; each R is independentlyhydrogen, an optionally substituted C₁₋₆ aliphatic group, or anoptionally substituted 3-8 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, wherein: two R on the same nitrogen atom areoptionally taken together with said nitrogen atom to form a 3-8 memberedsaturated, partially unsaturated, or aryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur; n is 0-2; R³,R⁴, R⁷, and R⁸ are each independently selected from halogen, R, OR, asuitably protected hydroxyl group, SR, a suitably protected thiol group,SO₂R, OSO₂R, N(R)₂, a suitably protected amino group, NR(CO)R,NR(CO)(CO)R, NR(CO)N(R)₂, NR(CO)OR, (CO)OR, O(CO)R, (CO)N(R)₂, orO(CO)N(R)₂; m is 0-2; R⁵ is T-C(R′)₃, T-C(R′)₂C(R″)₃, R, OR, a suitablyprotected hydroxyl group, SR, a suitably protected thiol group, SO₂R,OSO₂R, N(R)₂, a suitably protected amino group, NR(CO)R, NR(CO)(CO)R,NR(CO)N(R)₂, NR(CO)OR, (CO)OR, O(CO)R, (CO)N(R)₂, or O(CO)N(R)₂, or:each T is independently a valence bond or an optionally substitutedstraight or branched, saturated or unsaturated, C₁₋₆ alkylidene chainwherein up to two methylene units of T are optionally and independentlyreplaced by —O—, —N(R)—, —S—, —C(O)—, —S(O)—, or —S(O)₂—; each R′ and R″is independently selected from R, OR, SR, SO₂R, OSO₂R, N(R)₂, NR(CO)R,NR(CO)(CO)R, NR(CO)N(R)₂, NR(CO)OR, (CO)OR, O(CO)R, (CO)N(R)₂, orO(CO)N(R)₂; R⁹ and R^(9′) are each independently selected from halogen,R, OR, SR, or N(R)₂, or R¹ and R² are taken together to form a 3-7membered saturated, partially unsaturated, or aryl ring having 0-2heteroatoms independently selected from nitrogen, oxygen, or sulfur; Qis a valence bond or an optionally substituted straight or branched,saturated or unsaturated, C₁₋₆ alkylidene chain wherein up to twomethylene units of Q are optionally and independently replaced by —O—,—N(R)—, —S—, —C(O)—, —S(O)—, or —S(O)₂—; and R¹⁰ is R, a suitablyprotected hydroxyl group, a suitably protected thiol group, a suitablyprotected amino group, an optionally substituted 3-8 membered saturated,partially unsaturated, or aryl monocyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, an optionallysubstituted 8-10 membered saturated, partially unsaturated, or arylbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, a detectable moiety, a polymer residue, apeptide, or a sugar-containing or sugar-like moiety, comprising thesteps of: (a) providing a compound of formula II:

and (b) converting the compound of formula II to the compound of formulaI.
 2. The method according to claim 1, where the conversion at step (b)is achieved by intramolecular dehydration reaction.
 3. The methodaccording to claim 2, wherein the dehydration is performed in thepresence of a suitable acid.
 4. The method according to claim 3, whereinthe suitable acid is a Brønsted acid.
 5. The method according to claim4, wherein the Brønsted acid is a hydrogen halide, a carboxylic acid, asulfonic acid, or a phosphoric acid.
 6. The method according to claim 5,optionally performed in the presence of a suitable solvent.
 7. Themethod according to claim 6, wherein the suitable solvent is a proticsolvent, a halogenated hydrocarbon, an ether, an ester, an aromatichydrocarbon, a polar or a non-polar aprotic solvent, or any mixturesthereof.
 8. The method according to claim 1, wherein the compound offormula I is a compound of formula I-a:


9. The method according to claim 1, wherein the compound of formula I isa compound of formula I-b:


10. The method according to claim 1, wherein the compound of formula Iis a compound of formula I-c:


11. A method for preparing a compound of formula I-i:

or a pharmaceutically acceptable salt thereof, wherein: Q is a valencebond or an optionally substituted straight or branched, saturated orunsaturated, C₁₋₆ alkylidene chain wherein up to two methylene units ofQ are optionally and independently replaced by —O—, —N(R)—, —S—, —C(O)—,—S(O)—, or —S(O)₂—; R¹⁰ is R, a suitably protected hydroxyl group, asuitably protected thiol group, a suitably protected amino group, anoptionally substituted 3-8 membered saturated, partially unsaturated, oraryl monocyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, an optionally substituted 8-10 memberedsaturated, partially unsaturated, or aryl bicyclic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, adetectable moiety, a polymer residue, a peptide, or a sugar-containingor sugar-like moiety; and R is hydrogen, an optionally substituted C₁₋₆aliphatic group, or an optionally substituted 3-8 membered saturated,partially unsaturated, or aryl ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein: two R on the samenitrogen atom are optionally taken together with said nitrogen atom toform a 3-8 membered saturated, partially unsaturated, or aryl ringhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, comprising the steps of: (a) providing a compound of formulaI-ii:

and (b) converting compound I-ii to compound I-i.
 12. A method forpreparing a compound of formula I-iii:

or a pharmaceutically acceptable salt thereof, wherein: Q is a valencebond or an optionally substituted straight or branched, saturated orunsaturated, C₁₋₆ alkylidene chain wherein up to two methylene units ofQ are optionally and independently replaced by —O—, —N(R)—, —S—, —C(O)—,—S(O)—, or —S(O)₂—; R¹⁰ is R, a suitably protected hydroxyl group, asuitably protected thiol group, a suitably protected amino group, anoptionally substituted 3-8 membered saturated, partially unsaturated, oraryl monocyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, an optionally substituted 8-10 memberedsaturated, partially unsaturated, or aryl bicyclic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, adetectable moiety, a polymer residue, a peptide, or a sugar-containingor sugar-like moiety; and R is hydrogen, an optionally substituted C₁₋₆aliphatic group, or an optionally substituted 3-8 membered saturated,partially unsaturated, or aryl ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein: two R on the samenitrogen atom are optionally taken together with said nitrogen atom toform a 3-8 membered saturated, partially unsaturated, or aryl ringhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, comprising the steps of: (a) providing a compound of formulaI-iv:

and (b) converting compound I-iv to compound I-iii.
 13. The methodaccording to claim 1, wherein: G is O.
 14. The method according to claim1, wherein: R⁵ is T-C(R′)₃ or T-C(R′)₂C(R″)₃; each T is independently avalence bond or a straight or branched C₁₋₄ alkylidene chain wherein onemethylene unit of T is optionally replaced by —O—, —N(R)—, or —S—; andeach R′ and R″ is independently R, OR, OC(O)R, SR, or N(R)₂.
 15. Themethod according to claim 14, wherein: Q is an optionally substitutedstraight or branched, saturated or unsaturated, C₁₋₂, alkylidene chainwherein up to one methylene unit of Q is optionally replaced by —O—,—N(R)—, or —S—; and R¹⁰ is a glycoside.
 16. The method according toclaim 15, wherein Q is —O— and R¹⁰ is an arabinopyranoside or axylopyranoside.
 17. A composition comprising a compound of formula I,prepared according to claim 1, and a pharmaceutically acceptablecarrier, adjuvant, or vehicle.
 18. A method for inhibiting amyloid-betapeptide production in a patient, wherein said method comprisesadministering to said patient a composition according to claim
 17. 19. Amethod for inhibiting amyloid-beta (1-42) peptide production in apatient, wherein said method comprises administering to said patient acomposition according to claim
 17. 20. The method according to claim 19,wherein amyloid-beta (1-42) peptide levels are reduced and amyloid-beta(1-40) peptide levels are not substantially reduced. 21-26. (canceled)